The central focus of our laboratory is to understand the molecular mechanisms that control development of the three muscle cell types; skeletal, cardiac, and smooth. Members of the myocyte enhancer factor-2 (MEF2) family of transcription factors are the only known myogenic regulatory factors required for myogenesis and morphogenesis of all muscle cell types. These factors act combinatorially with other transcription factors to control muscle gene expression. In addition, recent studies have revealed important roles for MEF2 factors as transcriptional targets of calcium-dependent signaling pathways. Our results suggest that calcium-dependent signaling through MEF2 is important in cardiac hypertrophy and control of slow fiber gene expression in skeletal muscle. There are four MEF2 genes, MEF2A, -B, -C, and -D, in vertebrates and a single MEF2 gene in Drosophila. In Drosophila, embryos homozygous for a MEF2 null allele, skeletal, cardiac, and visceral myoblasts are specified, but there is a complete block in myoblast differentiation, revealing an essential role for MEF2 in myogenesis. One of the goals of the previous funding period was to create null mutations for the four MEF2 genes in mice and determine the consequences on muscle development. These studies have demonstrated unique roles for each of the MEF2 genes, but also revealed a significant degree of functional redundancy among the genes. The goals of this project represent a logical extension of our previous studies and are designed to further define the functions of the vertebrate MEF2 genes, to identify key downstream target genes for MEF2 factors, and to identify the regulatory factors and signaling systems that act upstream of MEF2 genes in each myogenic lineage. These studies will provide insights into fundamental mechanisms for muscle development and disease.